Anti-BCMA heavy chain-only antibodies

ABSTRACT

Anti-BCMA heavy chain-only antibodies (HCAb) and disclosed, along with methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to treat B-cell disorders characterized by the expression of BCMA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of the filing date of U.S.Provisional Patent Application Ser. No. 62/437,588, filed on Dec. 21,2016, the disclosure of which application is herein incorporated byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 17, 2018, isnamed TNO-0002-US_SL.txt and is 46 KB in size.

FIELD OF THE INVENTION

The present invention concerns anti-BCMA heavy chain-only antibodies(HCAb). The invention further concerns methods of making suchantibodies, compositions, including pharmaceutical compositions,comprising such antibodies, and their use to treat a B-cell disordercharacterized by the expression of BCMA.

BACKGROUND OF THE INVENTION

B-Cell Maturation Antigen (BCMA)

BCMA, also known as tumor necrosis factor superfamily member 17(TNFRSF17) (UniProt Q02223), is a cell surface receptor exclusivelyexpressed on plasma cells and plasmablasts. BCMA is a receptor for twoligands in the tumor necrosis factor (TNF) superfamily: APRIL (aproliferation-inducing ligand, also known as TNFSF13; TALL-2 and TRDL-1;the high affinity ligand for BCMA) and B cell activation factor (BAFF)(also known as BLyS; TALL-1; THANK; zTNF4; TNFSF20; and D8Ertd387e; thelow affinity ligand for BCMA). APRIL and BAFF are growth factors thatbind BCMA and promote survival of plasma cells. BCMA is also highlyexpressed on malignant plasma cells in human multiple myeloma (MM).Antibodies binding to BCMA are described, for example, in Gras et al.,1995, Int. Immunol. 7:1093-1106, WO200124811 and WO200124812. Anti-BCMAantibodies that cross-react with TACI are described in WO2002/066516.Bispecific antibodies against BCMA and CD3 are described, for example,in US 2013/0156769 A1 and US 2015/0376287 A1. An anti-BCMA antibody-MMAEor -MMAF conjugate has been reported to selectively induce killing ofmultiple myeloma cells (Tai et al., Blood 2014, 123(20): 3128-38). Aliet al., Blood 2016, 128(13):1688-700, have reported that in a clinicaltrial (#NCT02215967) chimeric antigen receptor (CAR) T cells targetingBCMA resulted in remission of multiple myeloma in human patients.

Heavy Chain-Only Antibodies

In a conventional IgG antibody, the association of the heavy chain andlight chain is due in part to a hydrophobic interaction between thelight chain constant region and the CH1 constant domain of the heavychain. There are additional residues in the heavy chain framework 2(FR2) and framework 4 (FR4) regions that also contribute to thishydrophobic interaction between the heavy and light chains.

It is known, however, that sera of camelids (sub-order Tylopoda whichincludes camels, dromedaries and llamas) contain a major type ofantibodies composed solely of paired H-chains (heavy-chain onlyantibodies or HCAbs). The HCAbs of Carnelidae (Camelus dromedarius,Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca and Lamavicugna) have a unique structure consisting of a single variable domain(VHH), a hinge region and two constant domains (CH2 and CH3), which arehighly homologous to the CH2 and CH3 domains of classical antibodies.These HCAbs lack the first domain of the constant region (CH1) which ispresent in the genome, but is spliced out during mRNA processing. Theabsence of the CH1 domain explains the absence of the light chain in theHCAbs, since this domain is the anchoring place for the constant domainof the light chain. Such HCAbs naturally evolved to conferantigen-binding specificity and high affinity by three CDRs fromconventional antibodies or fragments thereof (Muyldermans, 2001; JBiotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther5:111-124). Cartilaginous fish, such as sharks, have also evolved adistinctive type of immunoglobulin, designated as IgNAR, which lacks thelight polypeptide chains and is composed entirely by heavy chains. IgNARmolecules can be manipulated by molecular engineering to produce thevariable domain of a single heavy chain polypeptide (vNARs) (Nuttall etal. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function andBioinforrnatics 55, 187-197 (2004); Dooley et al., Molecular Immunology40, 25-33 (2003)).

The ability of heavy chain-only antibodies devoid of light chain to bindantigen was established in the 1960s (Jaton et al. (1968) Biochemistry,7, 4185-4195). Heavy chain immunoglobulin physically separated fromlight chain retained 80% of antigen-binding activity relative to thetetrameric antibody. Sitia et al. (1990) Cell, 60, 781-790 demonstratedthat removal of the CH1 domain from a rearranged mouse μ gene results inthe production of a heavy chain-only antibody, devoid of light chain, inmammalian cell culture. The antibodies produced retained VH bindingspecificity and effector functions.

Heavy chain antibodies with a high specificity and affinity can begenerated against a variety of antigens through immunization (van derLinden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) andthe VHH portion can be readily cloned and expressed in yeast (Frenken,L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels ofexpression, solubility and stability are significantly higher than thoseof classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBS Lett.414, 521-526 (1997)).

Mice in which the λ (lambda) light (L) chain locus and/or the λ and κ(kappa) L chain loci have been functionally silenced and antibodiesproduced by such mice are described in U.S. Pat. Nos. 7,541,513 and8,367,888. Recombinant production of heavy chain-only antibodies in miceand rats has been reported, for example, in WO2006008548; U.S.Application Publication No. 20100122358; Nguyen et al., 2003,Immunology; 109(1), 93-101; Brüggemann et al., Crit. Rev. Immunol.;2006, 26(5):377-90; and Zou et al., 2007, J Exp Med; 204(13): 3271-3283.The production of knockout rats via embryo microinjections ofzinc-finger nucleases is described in Geurts et al., 2009, Science,325(5939):433. Soluble heavy chain-only antibodies and transgenicrodents comprising a heterologous heavy chain locus producing suchantibodies are described in U.S. Pat. Nos. 8,883,150 and 9,365,655.CAR-T structures comprising single-domain antibodies as binding(targeting) domain are described, for example, in Iri-Sofia et al.,2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014,Biochim Biophys Acta, 1840:378-386.

SUMMARY OF THE INVENTION

The present invention concerns anti-BCMA antibodies.

In one aspect, the invention concerns a heavy chain-only antibodybinding to human B-Cell Maturation Antigen (BCMA) comprising a heavychain variable region comprising:

-   -   (a) a CDR1 having at least 95% sequence identity to any of the        sequences of SEQ ID NOs: 1 to 4; and/or    -   (b) a CDR2 having at least 95% sequence identity to any of the        sequences of SEQ ID NOs: 5 to 18; and/or    -   (c) a CDR3 having at least 95% sequence identity to SEQ ID NO:        19.

In one embodiment, the CDR1, CDR2, and CDR3 sequences are present in ahuman framework.

In another embodiment, the anti-BCMA heavy chain-only antibody furthercomprises a heavy chain constant region sequence in the absence of a CH1sequence.

In yet another embodiment, the heavy chain-only antibody comprises:

-   -   (a) a sequence selected from the group consisting of SEQ ID NOs:        1 to 4; and/or    -   (b) a CDR2 sequence selected from the group consisting of SEQ ID        NOs: 5 to 18; and/or    -   (c) a CDR3 of SEQ ID NO: 19.

In a further embodiment, the anti-BCMA heavy chain-only antibodycomprises:

-   -   (a) a CDR1 sequence selected from the group consisting of SEQ ID        NOs: 1 to 4; and    -   (b) a CDR2 sequence selected from the group consisting of SEQ ID        N Os: 5 to 18; and    -   (c) a CDR3 of SEQ ID NO: 19.

In a still further embodiment, the heavy chain-only anti-BCMA antibodycomprises a heavy chain variable region having at least 95% sequenceidentity to any of the sequences of SEQ ID NOs: 20 to 53.

In another embodiment, the heavy chain-only anti-BCMA antibody comprisesa heavy chain variable region sequence selected from the groupconsisting of SEQ ID NOs: 20 to 53.

In another aspect, the invention concerns a heavy chain-only antibodybinding to human B-Cell Maturation Antigen (BCMA) comprising a heavychain variable region comprising a heavy chain variable comprising

-   -   (a) a CDR1 sequence of the formula:        G F T F X1 X2Y A  (SEQ ID NO: 55)        where    -   X1 is S or T; and    -   X2 is S, N or R; and    -   (b) CDR2 sequence of the formula:        X3 X4 X5 X6 G X7 X8 X9  (SEQ ID NO: 56)        where    -   X3 is I or L;    -   X4 is S, T, I or V;    -   X5 is G or E;    -   X6 is S, G, N or D;    -   X7 is G, D or A;    -   X8 is S, Tor N;    -   X9 is T or S, and    -   (c) a CDR3 sequence of SEQ ID NO: 19    -   in a human VH framework.

In a further aspect, the invention concerns a heavy chain-only antibodybinding to human B-Cell Maturation Antigen (BCMA) comprising a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences in ahuman VH framework wherein the CDR sequences are a sequence with atleast 95% identity to a CDR sequence selected from the group consistingof SEQ ID NOs:1-19.

In one embodiment, such antibody comprises a heavy chain variable regioncomprising CDR1, CDR2 and CDR3 sequences in a human VH framework whereinthe CDR sequences are selected from the group consisting of SEQ IDNOs:1-19.

In a further aspect, the invention concerns a heavy chain-only antibodybinding to human B-Cell Maturation Antigen (BCMA) comprising a heavychain variable region comprising

-   -   (a) a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 5; and a CDR3        of SEQ ID NO: 19; or    -   (b) a CDR1 of SEQ ID NO: 2, a CDR2 of SEQ ID NO: 5; and a CDR3        of SEQ ID NO: 19 in a human VH framework.

In one embodiment, the heavy chain-only antibody is multi-specific, suchas bispecific.

In another embodiment, the heavy chain-only antibody has bindingaffinity to two different BCMA proteins.

In yet another embodiment, the heavy chain-only antibody has bindingaffinity to two different epitopes on the same BCMA protein.

In a further embodiment, the heavy chain-only antibody has bindingaffinity to an effector cell.

In a still further embodiment, the heavy chain-only antibody has bindingaffinity to a T-cell antigen.

In another embodiment, the heavy chain-only antibody has bindingaffinity to CD3.

In a different aspect, the heavy chain-only antibody hereinabovedescribed is in a CAR-T format.

In a further aspect, the invention concerns a pharmaceutical compositioncomprising an anti-BCMA heavy chain-only antibody herein.

In a still further aspect, the invention concerns a method for thetreatment of a B-cell disorder characterized by the expression of BCMAcomprising administering to a subject with such disorder an anti-BCMAheavy chain-only antibody herein, or a pharmaceutical compositioncomprising such antibody.

In various embodiments, the B-cell disorder may, for example, bemultiple myeloma or systemic lupus erythematosus.

In a further aspect, the invention concerns a polynucleotide encoding ananti-BCMA heavy chain-only antibody herein.

In a still further aspect, the invention concerns a vector comprising apolynucleotide encoding an anti-BCMA heavy chain-only antibody herein.

In another aspect, the invention concerns a cell comprising apolynucleotide encoding an anti-BCMA heavy chain-only antibody herein,or a vector comprising such polynucleotide.

In yet another aspect, the invention concerns a method of producing anantibody herein, comprising growing a cell as hereinabove describedunder conditions permissive for expression of the antibody, andisolating the antibody from the cells.

In a further aspect, the invention concerns a method of making ananti-BCMA heavy chain-only antibody as described herein, comprisingimmunizing a UniRat animal with BCMA and identifying BCMA-binding heavychain sequences.

These and further aspects will be further explained in the rest of thedisclosure, including the Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the CDR1, CDR2 and CDR3 amino acid sequences of 34 heavychain-only anti-BCMA antibodies of the invention.

FIG. 2 shows the heavy chain variable region sequences of 34 heavychain-only anti-BCMA antibodies of the invention.

FIG. 3: Heatmap summary of the antibody binding activities of 34 heavychain-only anti-BCMA antibodies expressed in HEK 293 cells. Redindicates strong binding while blue represents weak or negative bindingresults. Cell binding assays were run on all 34 antibodies and wereshown to bind H929 cells. Two antibodies encoding either VH sequence308607 or VH sequence 308915 were tested for binding to an untaggedhuman BCMA ECD fragment, and two off-target protein controls includinghuman IgG1 and a baculovirus protein extract (BVP), and BCMA+MM cellline RPMI8226. Two UniAbs were also evaluated for the ability to blockAPRIL (ligand)/BCMA (receptor) binding in a recombinant proteinELISA-style assay.

FIG. 4: Serum binding activity against the immunogen and other on- andoff-target proteins. A single 1:500 dilution of d35 scrum from a subsetof the animals was assayed for binding activity to a series of proteinsin a standard ELISA assay. On-target proteins include human BCMA Fcregion (huBCMA-Fc), a cyno BCMA ECD fused to a human Fc sequence(cyBCMA), and huBCMA from two other sources that do not have the Fcfusion (E. coli and wheat germ). Human serum albumin and human IgG1 wereused to assess off-target reactivity.

FIG. 5: Serum binding activity against a BCMA+ cell line (NCI-H929).Titers were evaluated by testing for serum binding to NCI-H929 cells byflow cytometry using a PE-conjugated anti-rat IgG2a antibody. Percent ofNCI-H929 cells showing significant staining was assessed.

FIG. 6: Representative CAR-T constructs comprising a human VHextracellular binding domain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., ed., 1994); “A Practical Guide to MolecularCloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual”(Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies: ALaboratory Manual: Portable Protocol No. I, Cold Spring HarborLaboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory; (1988).

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Unless indicated otherwise, antibody residues herein are numberedaccording to the Kabat numbering system (e.g., Kabat et al., Sequencesof Immunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

All references cited throughout the disclosure, including patentapplications and publications, are incorporated by reference herein intheir entirety.

I. Definitions

By “comprising” it is meant that the recited elements are required inthe composition/method/kit, but other elements may be included to formthe composition/method/kit etc. within the scope of the claim.

By “consisting essentially of”, it is meant a limitation of the scope ofcomposition or method described to the specified materials or steps thatdo not materially affect the basic and novel characteristic(s) of thesubject invention.

By “consisting of”, it is meant the exclusion from the composition,method, or kit of any element, step, or ingredient not specified in theclaim.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The terms “heavy chain-only antibody,” “heavy-chain antibody” and “HCAb”are used interchangeably, and refer, in the broadest sense, toantibodies lacking the light chain of a conventional antibody. Since thehomodimeric HCAbs lack a light chain and thus a VL domain, the antigenis recognized by one single domain, i.e., the variable domain of theheavy chain of a heavy-chain antibody (VH). The term specificallyincludes, without limitation, homodimeric antibodies comprising the VHantigen-binding domain and the CH2 and CH3 constant domains, in theabsence of the CH1 domain; functional (antigen-binding) variants of suchantibodies, soluble VH variants, Tg-NAR comprising a homodimer of onevariable domain (V-NAR) and five C-like constant domains (C-NAR) andfunctional fragments thereof; and soluble single domain antibodies(sdAbs). In one embodiment, the heavy chain-only antibody is composed ofthe variable region antigen-binding domain composed of framework 1,CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In oneembodiment, the heavy chain-only antibody is composed of anantigen-binding domain, at least part of a hinge region and CH2 and CH3domains. In another embodiment, the heavy chain-only antibody iscomposed of an antigen-binding domain, at least part of a hinge regionand a CH2 domain. In a further embodiment, the heavy chain-only antibodyis composed of an antigen-binding domain, at least part of a hingeregion and a CH3 domain. Heavy chain-only antibodies in which the CH2and/or CH3 domain is truncated are also included herein. In a furtherembodiment the heavy chain is composed of an antigen binding domain, andat least one CH (CH1, CH2, CH3, or CH4) domain but no hinge region. Theheavy chain-only antibody can be in the form of a dimer, in which twoheavy chains are disulfide bonded other otherwise, covalently ornon-covalently attached with each other. The heavy chain-only antibodymay belong to the IgG subclass, but antibodies belonging to othersubclasses, such as IgM, IgA, IgD and IgE subclass, are also includedherein. In a particular embodiment, the heavy chain antibody is of theIgG1, IgG2, IgG3, or IgG4 subtype, in particular IgG1 subtype. In oneembodiment, the heavy chain-only antibodies herein are used as a binding(targeting) domain of a chimeric antigen receptor (CAR).

The term “variable”, as used in connection with antibodies, refers tothe fact that certain portions of the antibody variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular antigen.However, the variability is not evenly distributed throughout thevariable domains of antibodies. It is concentrated in three segmentscalled hypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FRs). The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (sec Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 31-35 (H1),50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991))and/or those residues from a “hypervariable loop” residues 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothiaand Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework Region” or “FR”residues are those variable domain residues other than the hypervariableregion residues as herein defined.

Exemplary CDR designations are shown herein, however one of skill in theart will understand that a number of definitions of the CDRs arecommonly in use, including the Kabat definition (see “Zhao et al. Agermline knowledge based computational approach for determining antibodycomplementarity determining regions.” Mol Immunol. 2010; 47:694-700),which is based on sequence variability and is the most commonly used.The Chothia definition is based on the location of the structural loopregions (Chothia et al. “Conformations of immunoglobulin hypervariableregions.” Nature. 1989; 342:877-883). Alternative CDR definitions ofinterest include, without limitation, those disclosed by Honegger, “Yetanother numbering scheme for immunoglobulin variable domains: anautomatic modeling and analysis tool.” J Mol Biol. 2001; 309:657-670;Ofran et al. “Automated identification of complementarity determiningregions (CDRs) reveals peculiar characteristics of CDRs and B cellepitopes.” J Immunol. 2008; 181:6230-6235; Almagro “Identification ofdifferences in the specificity-determining residues of antibodies thatrecognize antigens of different size: implications for the rationaldesign of antibody repertoires.” J Mol Recognit. 2004; 17:132-143; andPadlan et al. “Identification of specificity-determining residues inantibodies.” Faseb J. 1995; 9:133-139., each of which is hereinspecifically incorporated by reference.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaccous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

Antibodies of the invention include multi-specific antibodies.Multi-specific antibodies have more than one binding specificity. Theterm “multi-specific” specifically includes “bispecific” and“trispecific,” as well as higher-order independent specific bindingaffinities, such as higher-order polyepitopic specificity, as well astetravalent antibodies and antibody fragments. “Multi-specific”antibodies specifically include antibodies comprising a combination ofdifferent binding entities as well as antibodies comprising more thanone of the same binding entity. The terms “multi-specific antibody,”multi-specific single chain-only antibody” and “multi-specific HCAb” areused herein in the broadest sense and cover all antibodies with morethan one binding specificity.

The term “valent” as used herein refers to a specified number of bindingsites in an antibody molecule.

A “multi-valent” antibody has two or more binding sites. Thus, the terms“bivalent”, “trivalent”, and “tetravalent” refers to the presence of twobinding sites, three binding sites, and four binding sites,respectively. Thus, a bispecific antibody according to the invention isat least bivalent and may be trivalent, tetravalent, or otherwisemulti-valent.

A large variety of methods and protein configurations are known and usedfor the preparation of bispecific monoclonal antibodies (BsMAB),tri-specific antibodies, and the like.

The term “bispecific three-chain antibody like molecule” or “TCA” isused herein to refer to antibody-like molecules comprising, consistingessentially of, or consisting of three polypeptide subunits, two ofwhich comprise, consist essentially of, or consist of one heavy and onelight chain of a monoclonal antibody, or functional antigen-bindingfragments of such antibody chains, comprising an antigen-binding regionand at least one CH domain. This heavy chain/light chain pair hasbinding specificity for a first antigen. The third polypeptide subunitcomprises, consists essentially of, or consists of a heavy chain onlyantibody comprising an Fc portion comprising CH2 and/or CH3 and/or CH4domains, in the absence of a CH1 domain, and an antigen binding domainthat binds an epitope of a second antigen or a different epitope of thefirst antigen, where such binding domain is derived from or has sequenceidentity with the variable region of an antibody heavy or light chain.Parts of such variable region may be encoded by V_(H) and/or V_(L) genesegments, D and J_(H), gene segments, or J_(L) gene segments. Thevariable region may be encoded by rearranged V_(H)DJ_(H), V_(L)DJ_(H),V_(L)J_(L), or V_(L)J_(L) gene segments. A TCA protein makes use of aheavy chain-only antibody as hereinabove defined.

The term “chimeric antigen receptor” or “CAR” is used herein in thebroadest sense to refer to an engineered receptor, which grafts adesired binding specificity (e.g. the antigen-binding region of amonoclonal antibody or other ligand) to membrane-spanning andintracellular-signaling domains. Typically, the receptor is used tograft the specificity of a monoclonal antibody onto a T cell to create achimeric antigen receptos (CAR). (J Natl Cancer Inst, 2015;108(7):dvj439; and Jackson et al., Nature Reviews Clinical Oncology,2016; 13:370-383.) A representative CAR-T construct comprising a humanVH extracellular binding domain is shown in FIG. 6.

By “human idiotype” is meant a polypeptide sequence epitope present on ahuman antibody in the immunoglobulin heavy and/or light chain variableregion. The term “human idiotype” as used herein includes both naturallyoccurring sequences of a human antibody, as well as synthetic sequencessubstantially identical to the polypeptide found in naturally occurringhuman antibodies. By “substantially” is meant that the degree of aminoacid sequence identity is at least about 85%-95%. Preferably, the degreeof amino acid sequence identity is greater than 90%, more preferablygreater than 95%.

By a “chimeric antibody” or a “chimeric immunoglobulin” is meant animmunoglobulin molecule comprising amino acid sequences from at leasttwo different Ig loci, e.g., a transgenic antibody comprising a portionencoded by a human Ig locus and a portion encoded by a rat Ig locus.Chimeric antibodies include transgenic antibodies with non-humanFe-regions or artificial Fc-regions, and human idiotypes. Suchimmunoglobulins can be isolated from animals of the invention that havebeen engineered to produce such chimeric antibodies.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include Clq binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoictic cells in summarized is Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (Clq) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

“Binding affinity” refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art. Low-affinity antibodies generally bind antigen slowlyand tend to dissociate readily, whereas high-affinity antibodiesgenerally bind antigen faster and tend to remain bound.

As used herein, the “Kd” or “Kd value” refers to a dissociation constantdetermined by BioLayer Interferometry, using an Octet QK384 instrument(Fortebio Inc., Menlo Park, Calif.) in kinetics mode. For example,anti-mouse Fc sensors are loaded with mouse-Fc fused antigen and thendipped into antibody-containing wells to measure concentration dependentassociation rates (kon). Antibody dissociation rates (koff) are measuredin the final step, where the sensors are dipped into wells containingbuffer only. The Kd is the ratio of koff/kon. (For further details see,Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800,2009).

An “epitope” is the site on the surface of an antigen molecule to whicha single antibody molecule binds. Generally an antigen has several ormany different epitopes and reacts with many different antibodies. Theterm specifically includes linear epitopes and conformational epitopes.

“Epitope mapping” is the process of identifying the binding sites, orepitopes, of antibodies on their target antigens. Antibody epitopes maybe linear epitopes or conformational epitopes. Linear epitopes areformed by a continuous sequence of amino acids in a protein.Conformational epitopes are formed of amino acids that are discontinuousin the protein sequence, but which are brought together upon folding ofthe protein into its three-dimensional structure.

“Polyepitopic specificity” refers to the ability to specifically bind totwo or more different epitopes on the same or different target(s).

An antibody binds “essentially the same epitope” as a referenceantibody, when the two antibodies recognize identical or stericallyoverlapping epitopes. The most widely used and rapid methods fordetermining whether two epitopes bind to identical or stericallyoverlapping epitopes are competition assays, which can be configured inall number of different formats, using either labeled antigen or labeledantibody. Usually, the antigen is immobilized on a 96-well plate, andthe ability of unlabeled antibodies to block the binding of labeledantibodies is measured using radioactive or enzyme labels.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; or (c)relieving the disease, i.e., causing regression of the disease. Thetherapeutic agent may be administered before, during or after the onsetof disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy may be administered during the symptomatic stage ofthe disease, and in some cases after the symptomatic stage of thedisease.

A “therapeutically effective amount” is intended for an amount of activeagent which is necessary to impart therapeutic benefit to a subject. Forexample, a “therapeutically effective amount” is an amount whichinduces, ameliorates or otherwise causes an improvement in thepathological symptoms, disease progression or physiological conditionsassociated with a disease or which improves resistance to a disorder.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” encompass, without limitation,individuals having cancer, individuals with autoimmune diseases, withpathogen infections, and the like. Subjects may be human, but alsoinclude other mammals, particularly those mammals useful as laboratorymodels for human disease, e.g. mouse, rat, etc.

II. Detailed Description

Anti-BCMA Antibodies

The present invention provides a family of closely related heavychain-only antibodies that bind to human BCMA. The antibodies of thisfamily comprise a set of CDR sequences as defined herein and shown inFIG. 1, and are exemplified by the provided heavy chain variable region(VH) sequences of SEQ ID NOs 20 to 53 set forth in FIG. 2. The familiesof antibodies provide a number of benefits that contribute to utility asclinically therapeutic agent(s). The antibodies include members with arange of binding affinities, allowing the selection of a specificsequence with a desired binding affinity.

A suitable antibody may be selected from those provided herein fordevelopment and therapeutic or other use, including without limitationuse as a bispecific or tri-specific antibody, or part of a CAR-Tstructure. Determination of affinity for a candidate protein can beperformed using methods known in the art, such as Biacore measurements.Members of the antibody family may have an affinity for BCMA with a Kdof from about 10⁻⁶ to around about 10⁻¹¹, including without limitation:from about 10⁻⁶ to around about 10⁻¹⁰; from about 10⁻⁶ to around about10⁻⁹; from about 10⁻⁶ to around about 10⁻⁸; from about 10⁻⁸ to aroundabout 10⁻¹¹; from about 10⁻⁸ to around about 10⁻¹⁰; from about 10⁻⁸ toaround about 10⁻⁹; from about 10⁻⁹ to around about 10⁻¹¹; from about10⁻⁹ to around about 10⁻¹⁰; or any value within these ranges. Theaffinity selection may be confirmed with a biological assessment formodulating, e.g. blocking, a BCMA biological activity, including invitro assays, pre-clinical models, and clinical trials, as well asassessment of potential toxicity.

Members of the antibody family herein are not cross-reactive with theBCMA protein of Cynomolgus macaque but can be engineered to providecross-reactivity if desired.

The family of BCMA specific antibodies herein comprises a VH domain,comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. TheCDR sequences may be situated, as an example, in the region of aroundamino acid residues 26-35; 53-59; and 98-117 for CDR1, CDR2 and CDR3,respectively, of the provided exemplary variable region sequences setforth in SEQ ID NO: 20-53. It will be understood by one of skill in theart that the CDR sequences may be in different position if a differentframework sequence is selected, although generally the order of thesequences will remain the same.

The antibodies of the present invention share the CDR3 amino acidsequence of SEQ ID NO: 19. The CDR1 and CDR2 sequences of the anti-BCMAantibodies of the present invention may be encompassed by the followingstructural formulas, where an X indicates a variable amino acid, whichmay be specific amino acids as indicated below.CDR1G F T F X1 X2 Y A  (SEQ ID NO: 55)where

-   -   X1 is S or T;    -   X2 is S, N or R        CDR2        X3 X4 X5 X6 G X7 X8 X9  (SEQ ID NO: 56)        where    -   X3 is I or L;    -   X4 is S, T, I or V;    -   X5 is G or E;    -   X6 is S, G, N or D;    -   X7 is G, D or A;    -   X8 is S, Tor N;    -   X9 is T or S.

Representative CDR1, CDR2, and CDR3 sequences are shown in FIG. 1.

In some embodiments the anti-BCMA antibody of the invention comprises aCDR1 sequence of SEQ ID NO: 1, 2, 3, or 4. In a particular embodiment,the CDR1 sequence is SEQ ID NO: 1 or SEQ ID NO: 2.

In some other embodiments the anti-BCMA antibody of the inventioncomprises a CDR2 sequence of any one of SEQ ID NOs: 5-18. In aparticular embodiment, the CDR2 sequence is SEQ ID NO: 5.

In another embodiment, the anti-BCMA antibody of the present inventioncomprises a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO:5 and a CDR3 sequence of SEQ ID NO: 19.

In a further embodiment, the anti-BCMA antibody of the present inventioncomprises a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO:5 and a CDR3 sequence of SEQ ID NO: 19.

In further embodiments, the anti-BCMA antibody of the present inventioncomprises any of the heavy chain amino acid sequences of SEQ ID NOs: 20to 53 (FIG. 2).

In some embodiments a CDR sequence of the invention comprises one, two,three or more amino acid substitutions relative to a CDR1, CDR2 and/orCDR3 sequence or set of CDR1, CDR2 and CDR3 sequences in any one of SEQID NOs:1 to 19 (FIG. 1). In some embodiments said amino acidsubstitution(s) are one or more of amino acid positions 5 and 6 of CDR1,and/or one or more of the amino acid positions of 1-4 and 6-8 of CDR2,relative to the formulas provided above. In some embodiments the CDRsequences of an anti-BCMA antibody herein have a sequence with at least85% identity, at least 90% identity, at least 95% identity, at least 98%identify, or at least 99% identity relative to a CDR1, CDR2 and/or CDR3sequence or set of CDR1, CDR2, and CDR3 sequences in any one of SEQ IDNOs: 1 to 19 (FIG. 1). In some other embodiments, the single chain-onlyanti-BCMA antibodies herein will comprise a heavy chain variable regionsequence with at least 85% identity, at least 90% identity, at least 95%identity, at least 98% identity, or at least 99% identity to the heavychain variable region sequences shown in FIG. 2.

In some embodiments, bispecific or multispecific antibodies areprovided, which may have any of the configurations discussed herein,including without limitation a three chain bispecific antibody.Bispecific antibodies comprise at least the heavy chain variable regionof an antibody specific for a protein other than BCMA.

Where a protein of the invention is a bispecific antibody, one bindingmoiety is specific for human BCMA while the other arm may be specificfor target cells, tumor associated antigens, targeting antigens, e.g.integrins, etc., pathogen antigens, checkpoint proteins, and the like.Target cells specifically include cancer cells, such as hematologictumors, e.g. B-cell tumors.

Various formats of bispecific antibodies are within the ambit of theinvention, including without limitation single chain polypeptides, twochain polypeptides, three chain polypeptides, four chain polypeptides,and multiples thereof. The bispecific antibodies herein specificallyinclude T-cell bispecific antibodies binding to BCMA, which isselectively expressed on plasma cells (PCs) and multiple myeloma (MM),and CD3 (anti-BCMA×anti-CD3 antibodies). Such antibodies induce potentT-cell mediated killing of cells carrying BCMA.

Pharmaceutical Compositions

It is another aspect of the present invention to provide pharmaceuticalcompositions comprising one or more antibodies of the present inventionin admixture with a suitable pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers as used herein are exemplified, butnot limited to, adjuvants, solid carriers, water, buffers, or othercarriers used in the art to hold therapeutic components, or combinationsthereof.

Pharmaceutical composition of the antibodies used in accordance with thepresent invention are prepared for storage by mixing proteins having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (see, e.g. Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), such as inthe form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Anti-BCMA antibody formulations are disclosed, for example, in U.S. Pat.No. 9,034,324. Similar formulations can be used for the proteins of thepresent invention.

Methods of Use

The pharmaceutical compositions herein can be used for the treatment ofB-cell related disorders characterized by the expression of BCMA.

Such B-cell related disorders include B-cell and plasma cellmalignancies and autoimmune disorders, including, without limitation,plasmacytoma, Hodgkins' lymphoma, follicular lymphomas, smallnon-cleaved cell lymphomas, endemic Burkitt's lymphoma, sporadicBurkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associatedlymphoid tissue lymphoma, nodal monocytoid B cell lymphoma, spleniclymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed celllymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma,pulmonary B cell angiocentric lymphoma, small lymphocytic lymphoma, Bcell proliferations of uncertain malignant potential, lymphomatoidgranulomatosis, post-transplant lymphoproliferative disorder, animmunoregulatory disorder, rheumatoid arthritis, myasthenia gravis,idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chagas'disease, Grave's disease, Wegener's granulomatosis, poly-arteritisnodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiplesclerosis, anti-phospholipid syndrome, ANCA associated vasculitis,Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia,and rapidly progressive glomerulonephritis, heavy-chain disease, primaryor immunocyte-associated amyloidosis, or monoclonal gammopathy.

The plasma cell disorders characterized by the expression of BCMAinclude Multiple Myeloma (MM). MM is a B-cell malignancy characterizedby a monoclonal expansion and accumulation of abnormal plasma cells inthe bone marrow compartment. Current therapies for MM often causeremissions, but nearly all patients eventually relapse and die. There issubstantial evidence of an immune-mediated elimination of myeloma cellsin the setting of allogeneic hematopoietic stem cell transplantation;however, the toxicity of this approach is high, and few patients arecured. Although some monoclonal antibodies have shown promise fortreating MM in preclinical studies and early clinical trials, consistentclinical efficacy of any monoclonal antibody therapy for MM has not beenconclusively demonstrated. There is therefore a great need for newtherapies, including immunotherapies for MM (see, e.g. Carpenter et al.,Chin Cancer Res 2013, 19(8):2048-2060).

Another B-cell disorder involving plasma cells i.e. expressing BCMA issystemic lupus erythematosus (SLE), also known as lupus. SLE is asystemic, autoimmune disease that can affect any part of the body and isrepresented with the immune system attacking the body's own cells andtissue, resulting in chronic inflammation and tissue damage. It is aType III hypersensitivity reaction in which antibody-immune complexesprecipitate and cause a further immune response (Inaki & Lee, Nat RevRheumatol 2010; 6: 326-337).

The antibodies of the present invention can be used to developtherapeutic agents for the treatment of MM, SLE, and other B-celldisorders characterized by the expression of BCMA, such as those listedabove.

In one embodiment, the antibodies herein can be in the form of heavychain-only anti-BCMA antibody-CAR structures, i.e. heavy chain-onlyanti-BCMA antibody-CAR-transduced T cell structures.

Effective doses of the compositions of the present invention for thetreatment of disease vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human, but nonhuman mammals mayalso be treated, e.g. companion animals such as dogs, cats, horses,etc., laboratory mammals such as rabbits, mice, rats, etc., and thelike. Treatment dosages can be titrated to optimize safety and efficacy.

Dosage levels can be readily determined by the ordinarily skilledclinician, and can be modified as required, e.g., as required to modifya subject's response to therapy. The amount of active ingredient thatcan be combined with the carrier materials to produce a single dosageform varies depending upon the host treated and the particular mode ofadministration. Dosage unit forms generally contain between from about 1mg to about 500 mg of an active ingredient.

In some embodiments, the therapeutic dosage the agent may range fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. An exemplary treatmentregime entails administration once every two weeks or once a month oronce every 3 to 6 months. Therapeutic entities of the present inventionare usually administered on multiple occasions. Intervals between singledosages can be weekly, monthly or yearly. Intervals can also beirregular as indicated by measuring blood levels of the therapeuticentity in the patient. Alternatively, therapeutic entities of thepresent invention can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the polypeptidein the patient.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the antibodies and antibody structures described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the LD₅₀ (the dose lethal to50% of the population) or the LD₁₀₀ (the dose lethal to 100% of thepopulation). The dose ratio between toxic and therapeutic effect is thetherapeutic index. The data obtained from these cell culture assays andanimal studies can be used in formulating a dosage range that is nottoxic for use in humans. The dosage of the antibodies described hereinlies preferably within a range of circulating concentrations thatinclude the effective dose with little or no toxicity. The dosage canvary within this range depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition.

The compositions for administration will commonly comprise an antibodyor other ablative agent dissolved in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., buffered saline and the like. These solutions aresterile and generally free of undesirable matter. These compositions maybe sterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, e.g., sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate and the like. The concentration of active agentin these formulations can vary widely, and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thepatient's needs (e.g., Remington's Pharmaceutical Science (15th ed.,1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics(Hardman et al., eds., 1996)).

Also within the scope of the invention are kits comprising the activeagents and formulations thereof, of the invention and instructions foruse. The kit can further contain a least one additional reagent, e.g. achemotherapeutic drug, etc. Kits typically include a label indicatingthe intended use of the contents of the kit. The term label includes anywriting, or recorded material supplied on or with the kit, or whichotherwise accompanies the kit.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXAMPLES Example 1

Genetically Engineered Rats Expressing Heavy Chain-Only Antibodies

A ‘human-rat’ IgH locus was constructed and assembled in several parts.This involved the modification and joining of rat C region genesdownstream of human J_(H)s and subsequently, the upstream addition ofthe human V_(H)6-D-segment region. Two BACs with separate clusters ofhuman V_(H) genes [BAC6 and BAC3] were then co-injected with the BACtermed Georg, encoding the assembled and modified region comprisinghuman V_(H)6, all Ds, all J_(H)s, and modified rat Cγ2a/1/2b (ΔC_(H)1).

Transgenic rats carrying artificial heavy chain immunoglobulin loci inunrearranged configuration were generated. The IgG2a(ΔC_(H)1).,igG1(ΔC_(H)1)., IgG2b(ΔC_(H)1) genes lacked the C_(H)1 segment. Theconstant region genes IgE, IgA and 3′ enhancer were included in GeorgBAC. RT-PCR and serum analysis (ELISA) of transgenic rats revealedproductive rearrangement of transgenic immunoglobulin loci andexpression of heavy chain only antibodies of various isotypes in serum.Transgenic rats were cross-bred with rats with mutated endogenous heavychain and light chain loci previously described in US patent publication2009/0098134 A1. Analysis of such animals demonstrated inactivation ofrat immunoglobulin heavy and light chain expression and high levelexpression of heavy chain antibodies with variable regions encoded byhuman V, D, and J genes Immunization of transgenic rats resulted inproduction of high titer serum responses of antigen-specific heavy chainantibodies. These transgenic rats expressing heavy chain antibodies witha human VDJ region were called UniRats.

Example 2

Immunization

Immunization with Recombinant Extracellular Domain of BCMA.

Twelve UniRat animals (6 HC27, 6 HC28) were immunized with recombinanthuman BCMA protein. The animals were immunized according to standardprotocol using a Titermax/Alhydrogel adjuvant. Recombinant extracellulardomain of BCMA was purchased from R&D Systems and was diluted withsterile saline and combined with adjuvant. The immunogen was combinedwith Titermax and Alhydrogel adjuvants. The first immunization (priming)with immunogen in Titermax was administered in the left and right legs.Subsequent boosting immunizations were done in the presence ofAlhydrogel and three days before harvest boosts were performed withimmunogens in PBS. Serum was collected from rats at the final bleed todetermine serum titers.

Serum Titer Results

Serum titer summary information is shown in FIGS. 4 and 5. For half ofthe animals, binding activity for a single 1:500 serum titer dilutionwas tested by ELISA against a huBCMA+Fc protein and a cynoBCMA+Fcprotein produced in cukaryotic cells and two human BCMA proteins from E.coli and wheat germ, respectively. In addition, serum samples weretested against two off-target proteins, HSA and human IgG1. In addition,serum from all 12 animals was assayed for binding to NCI-H929 cells(BCMA+, lambda−).

Among this group of animals, we observed a significant spread in serumreactivity levels to NCI-H929 cells (BCMA+, lambda−). The relevance ofthese results is confirmed by the ELISA binding data generated for asubset of the animals. Some general Fc-specific reactivity is observedfor a number of the animals, but those that look the most promising alsoshow high apparent titer in the assays against untagged human BCMA fromalternative sources (E. coli, wheat germ) indicating significant BCMAECD-specific binding activity. Positive signal for binding to thecynoBCMA+Fc protein may reflect binding to either the ECD or the Fcportion of the molecule that is also included on the human immunogen. Inboth assay types, analysis of serum taken from these animals prior toimmunization showed no reactivity to the immunogen or off targetprotein.

Example 3

Gene Assembly, Expression and Binding Assays

309 cDNAs encoding heavy chain only antibodies highly expressed in lymphnode cells were selected for gene assembly and cloned into an expressionvector. Subsequently, these 309 heavy chain sequences were expressed inHEK cells as UniAb heavy chain only antibodies (CH1 deleted, no lightchain). Binding results including amino acid sequences for 34 VH regionstested can be found in the BCMA_FAM2_DATA.

Supernatants of two antibodies were tested for binding in a standardELISA assay to a human BCMA. Binding to recombinant BCMA protein wasdetermined by ELISA using human BCMA ECD obtained from Abcam (ab50089).The BCMA ECD protein was used at a concentration of 2 μg/mL to captureUniAbs at 50 ng/mL. Binding of UniAbs was detected with a goatanti-human IgG HRP conjugated antibody (ThermoFisher 31413). Allantibodies were diluted in 1×TBS with 0.05% Tween-20 and 1% dry milkpowder.

Off-target binding to Baculo Virus Protein Extract (BVP) was conductedby ELISA as above, with the modification of using 1×PBS and 1% dry milkpowder for the diluent. BVP extract was obtained from INSERM (Nantes,France). Binding to baculovirus particles (>5× over background in ourassay) is thought to indicate low-affinity interactions with humantissues which correlates with reduced half-lives of antibodies in humansand monkeys (Hotzcl et al., mAbs 4:6, p 753-760, 2012)._Off-targetbinding of human IgG1 was assessed by ELISA using the UniAbs to capturehuman IgG1 kappa followed by detection of the kappa chain with a goatanti-human kappa HRP conjugated antibody (Southern Biotech 2060-05).

Supernatants of all antibodies were also tested by flow cytometry forbinding to RPMI-8226 cells (BCMA+, lambda+) and H929 cells (BCMA+,lambda−). The samples were measured by flow cytometry using a GuavaeasyCyte 8HT instrument from EMD Millipore and analyzed using guavaSoft.Bound antibodies were detected with goat anti-human IgG F(ab′)₂conjugated to PE (Southern Biotech 2042-09). All antibodies were dilutedin PBS with 1% BSA. Positive staining was determined by comparison tostaining with a human IgG1 isotype control. The NCI-H929 and RPMI-8226cell lines are human multiple myeloma lines expressing human BCMA, whichwere obtained from the American Type Culture Collection (ATCC) andcultured according to ATCC recommendations.

Two UniAbs were also evaluated for the ability to block APRIL(ligand)/BCMA (receptor) binding in a recombinant protein ELISA-styleassay. To evaluate blocking of the receptor/ligand interaction betweenBCMA and APRIL, recombinant human BCMA (Sino Biological 10620-H03H) wasdirectly coated on plates followed by incubation with a dilution seriesof each UniAb. HA-tagged recombinant APRIL protein (RnD Systems5860-AP-010) was then incubated with the BCMA/antibody complexes andbinding of APRIL to BCMA was detected using a chicken anti-HA antibodyconjugated to HRP (Abcam ab1190). A RnD systems anti-BCMA antibody(AF193) was used as a positive control for BCMA/APRIL blocking.

An additional off-target binding assay was run on intact baculovirusparticles (BVPs) though none of the tested UniAbs showed positivebinding.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The invention claimed is:
 1. A heavy chain-only antibody binding tohuman B-Cell Maturation Antigen (BCMA) comprising a heavy chain variableregion comprising: (a) a CDR1 sequence comprising SEQ ID NO: 1, a CDR2sequence comprising SEQ ID NO: 5, and a CDR3 sequence comprising SEQ IDNO: 19; or (b) a CDR1 sequence comprising SEQ ID NO: 2, a CDR2 sequencecomprising SEQ ID NO: 54, and a CDR3 sequence comprising SEQ ID NO: 19;in a human VH framework.
 2. The heavy chain-only antibody of claim 1,further comprising a heavy chain constant region sequence in the absenceof a CH1 sequence.
 3. The heavy chain-only antibody of claim 1, which isin a CAR-T format.
 4. The heavy chain-only antibody of claim 1, which ispresent in a CAR-transduced T-cell.
 5. A pharmaceutical compositioncomprising the heavy chain-only antibody of claim
 1. 6. The heavychain-only antibody of claim 1, wherein the heavy chain variable regioncomprises a CDR1 sequence comprising SEQ ID NO: 1, a CDR2 sequencecomprising SEQ ID NO: 5, and a CDR3 sequence comprising SEQ ID NO: 19,in a human VH framework.
 7. The heavy chain-only antibody of claim 6,wherein the heavy chain variable region has at least 95% sequenceidentity to SEQ ID NO:
 20. 8. The heavy chain-only antibody of claim 7,wherein the heavy chain variable region comprises SEQ ID NO:
 20. 9. Theheavy chain-only antibody of claim 6, further comprising a heavy chainconstant region sequence in the absence of a CH1 sequence.
 10. The heavychain-only antibody of claim 6, which is in a CAR-T format.
 11. Theheavy chain-only antibody of claim 6, which is present in aCAR-transduced T-cell.
 12. The heavy chain-only antibody of claim 1,wherein the heavy chain variable region comprises a CDR1 sequencecomprising SEQ ID NO: 2, a CDR2 sequence comprising SEQ ID NO: 54, and aCDR3 sequence comprising SEQ ID NO: 19, in a human VH framework.
 13. Theheavy chain-only antibody of claim 12, wherein the heavy chain variableregion has at least 95% sequence identity to SEQ ID NO:
 21. 14. Theheavy chain-only antibody of claim 13, wherein the heavy chain variableregion comprises SEQ ID NO:
 21. 15. The heavy chain-only antibody ofclaim 12, further comprising a heavy chain constant region sequence inthe absence of a CH1 sequence.
 16. The heavy chain-only antibody ofclaim 12, which is in a CAR-T format.
 17. The heavy chain-only antibodyof claim 12, which is present in a CAR-transduced T-cell.